This is a competitive grant-renewal application, in which the overall goal is to understand the mechanisms of Type I IFN signaling in malignant cells. During the previous funding period, we identified several novel-IFN activated signaling cascades involving the CrkL-adapter and demonstrated that CrkL plays a critical role in Type I IFN signal transduction. We will now undertake studies to establish the functional roles of the different domains of CrkL in the context of its signaling capacity and define the contribution of distinct CrkL-cascades in the generation of the biological effects of IFNs. A major goal of this grant application is also to determine the mechanisms of regulation of Stat-serine phosphorylation. In addition to tyrosine phosphorylation, IFNalpha-dependent phosphorylation of Stat1 and Stat3 on serine 727 is required for transcriptional activation of target genes. Although it is well established that Jak-kinases regulate tyrosine phosphorylation of Stats, the IFNalpha-activated serine kinase that phosphorylates Stat-proteins is not known. The identification of such a serine kinase is an important outstanding issue, required to complete our understanding on the IFN-activated Jak-Stat pathway. Our data have established that a member of the protein kinase C-family of proteins, PKC-delta, is activated in a Type I IFN-dependent manner and regulates serine phosphorylation of Stat1. These findings, for the first time, provide the identity of the IFNalpha-activated Stat-serine kinase and form the basis for further studies. The objective of this section of this proposal is to define the mechanisms by which PKC-delta is activated by the Type I IFN receptor and interacts with Stat1, and possibly Stat3, to effect their phosphorylation on serine 727. Studies will be also performed to define the role of PKC-delta activation/Stat-serine phosphorylation in the induction of the antiproliferative, antiviral and immunomodulatory effects of Type I IFNs, as well as the induction of suppressive effects of IFNalpha on bone marrow leukemic progenitors from patients with chronic myelogenous leukemia. Altogether, this proposal will address important aspects of the mechanisms of Type I IFN-signal transduction and generation of antileukemic effects.